A fundamental need exists in the developing world for rapid, point-of-care (POC) tests to diagnose and treat infectious diseases. The presence of abnormal urine proteins is used for the noninvasive diagnosis of infections outside of the urinary tract, e.g., legionella pneumonia. Early in the HIV epidemic, investigation demonstrated that HIV infection could be recognized in urine by the presence of antibodies to common HIV antigens. Minimal studies have been done to detect HIV proteins (or their degradation products) within urine to provide urinary markers of HIV infection. Although modern liquid chromatography used in tandem with mass spectrometry (LC/MS-MS) has ultrasensitive detection ability, i.e. femtomolar, high throughput capacity to characterize urine proteomes, and demonstrated ability to identify HIV-1 proteins in complex mixtures, it has not been exploited to identify differential protein markers in urine from HIV-positive patients. The lack of this information limits our ability to assess HIV replication using urine. Our long-term goal is to develop rapid, POC diagnostic tests using urine as a noninvasive biofluid. The objective of this project is to determine the differential urinary composition of HIV-positive subjects and discover markers of HIV infection. The central hypothesis of the application is that the modern investigative techniques of LC/MS-MS will demonstrate a unique urinary biomarker in those with highly active (serum viral load >100,000 viral copies/mL) HIV infection due to the excretion of a virus-derived protein/protein fragment(s). The rationale for the proposed research is that, once a specific urinary biomarker(s) is known, then a simple, rapid POC test can be developed, and novel noninvasive methods can be used to diagnose and monitor HIV infection. We will test our central hypothesis with the following specific aim: Identify urinary viral proteins or protein degradation fragments that correlate with highly active systemic HIV replication. The working hypothesis for this aim is that, with modern techniques, HIV excretion and thereby systemic replication are distinguishable in urine. To discover such a biomarker(s), we will use LC-MS/MS and human/HIV protein databases to identify urinary proteins/protein degradation products in subjects with highly active HIV replication. The proposed work is innovative because it applies the latest investigative techniques of biomarker discovery to HIV infection. The expected outcome is to provide a noninvasively obtained biomarker(s) of HIV replication for screening and treatment monitoring. The positive impact of this work includes providing an alternative means of diagnosing HIV, e.g., in neonates with maternal antibodies, acutely infected individuals, or subjects vaccinated with a candidate HIV vaccine as well as monitoring HIV replication by noninvasive testing. The proposed research is significant because it provides the fundamental data necessary for rapid, POC noninvasive testing that will allow alternative means of diagnosing and monitoring treatment of HIV in the developing world. PUBLIC HEALTH RELEVANCE: This proposal aims to discover biomarkers for the noninvasive detection of HIV infection using urine as the biological sample fluid. Using urine would be valuable because urine can contain biomarkers from nonurinary tract sites and bypass the false positive serologic results from certain patient groups, e.g., those previously vaccinated with candidate HIV vaccines. Upon accomplishment of this project, we will have furthered the development of methods for biomarker discovery in urine, which can then be applied to other infections.